Liposomal phosphodiester, phosphorothioate, and P-ethoxy oligonucleotides

ABSTRACT

An improved delivery system for antisense oligonucleotides involves a liposomal composition, comprising a liposome which consists essentially of neutral phospholipids and an antisense oligonucleotide that is entrapped in the liposome and is selected from the group consisting of phosphodiester oligonucleotides, phosphorothioate oligonucleotides, and p-ethoxy oligonucleotides.

BACKGROUND OF THE INVENTION

The present invention relates to liposomal formulations of certainantisense oligonucleotides, specifically liposomal phosphodiester,phosphorothioate, and p-ethoxy oligonucleotides. The invention alsorelates to methods of making such formulations and methods of using suchformulations in medical therapy.

Antisense oligonucleotides (oligos), complementary to specific regionsof the target mRNA, have been used to inhibit the expression ofendogenous genes. When the antisense oligonucleotides bind to the targetmRNA, a DNA-RNA hybrid is formed. This hybrid formation inhibits thetranslation of the mRNA and, thus, the gene's expression of the protein.If the protein is essential for the survival of the cell, the inhibitionof its expression may lead to cell death. Therefore, antisenseoligonucleotides can be useful tools in anticancer and antiviraltherapies.

The main obstacles in using antisense oligonucleotides to inhibit geneexpression are cellular instability, low cellular uptake, and poorintracellular delivery. Natural phosphodiesters are not resistant tonuclease hydrolysis; thus high concentrations of antisenseoligonucleotides are needed before any inhibitory effect is observed.Modified phosphodiester analogs, such as phosphorothioates, have beenmade to overcome this nuclease hydrolysis problem, but they have notprovided a completely satisfactory solution to the problem.

The cellular uptake of antisense oligonucleotides is low. To solve thisproblem, physical techniques such as calcium-phosphate precipitation,DEAE-dextran mediation, or electroporation have been used to increasethe cellular uptake of oligonucleotides. These techniques are difficultto reproduce and are inapplicable in vivo. Cationic lipids, such asLipofectin, have also been used to deliver phosphodiester orphosphorothioate oligonucleotides. An electrostatic interaction isformed between the cationic lipids and the negatively chargedphosphodiester or phosphorothioate oligonucleotides, which results in acomplex that is then taken up by the target cells. Since these cationiclipids do not protect the oligonucleotides from nuclease digestion, theyare only useful in delivering the nuclease-resistant phosphorothioates,but not the nuclease-cleavable phosphodiesters.

Another modified phosphodiester (PD) analog that has been prepared isp-ethoxy (pE) oligos. The modifications of pE oligos are made in thephosphate backbone so that the modification will not interfere with thebinding of these oligos to the target mRNA. pE oligos are made by addingan ethyl group to the nonbridging oxygen atom of the phosphate backbone,thus rendering these oligos uncharged compounds. In spite of theirresistance to nucleases, the cellular uptake and intracellular deliveryof pE oligos are still poor because upon internalization, these oligosremain sequestered inside the endosomal/lysosomal vacuoles, impedingtheir access to the target mRNA.

There is a need for improved antisense compositions for use in treatmentof disease, and also a need for processes for making such improvedcompositions.

SUMMARY OF THE INVENTION

One embodiment of the present invention is a liposomal composition ofantisense oligonucleotides. The composition includes (a) a liposomewhich consists essentially of neutral phospholipids, and (b) anantisense oligonucleotide that is entrapped in the liposome and isselected from the group consisting of phosphodiester oligonucleotides,phosphorothioate oligonucleotides, and p-ethoxy oligonucleotides. Thephospholipids are preferably phosphatidylcholines. An especiallypreferred phospholipid is dioleoylphosphatidyl choline. When theantisense oligonucleotide is a phosphodiester oligonucleotide, thepreferred molar ratio of phospholipid to oligo is less than about3,000:1. When the antisense oligonucleotide is a phosphorothioateoligonucleotide, the preferred molar ratio of phospholipid to oligo isbetween about 10:1 and about 50:1. When the antisense oligonucleotide isa p-ethoxy oligonucleotide, the preferred molar ratio of phospholipid tooligo is between about 5:1 and about 100:1.

Another embodiment of the present invention is a method of inhibitingthe growth of tumor cells in mammals. The method comprises the step ofadministering to a mammalian subject having a tumor an amount of theabove-described composition that is effective to inhibit the growth oftumor cells.

Another embodiment of the present invention is a method of preparing theliposomal composition of antisense oligonucleotides. The methodcomprises the steps of (a) hydrating a lyophilized composition thatconsists essentially of neutral phospholipids and an antisenseoligonucleotide that is selected from the group consisting ofphosphodiester oligonucleotides, phosphorothioate oligonucleotides, andp-ethoxy oligonucleotides, thereby forming an aqueous suspension whichincludes free oligonucleotide and liposomes entrapping oligonucleotide;and (b) separating the free oligonucleotide from the liposomes bydialysis. In a preferred embodiment, the aqueous suspension is sonicatedbefore dialysis.

The compositions of the present invention constitute an improveddelivery system for antisense oligos, such as those used in anti-cancertherapy. In addition to minimizing nuclease hydrolysis of the oligos,the liposomal compositions of the present invention result in increasedcellular uptake and intracellular delivery of the antisense oligos, ascompared to prior art compositions. Therefore, when such compositionsare used to deliver oligos that inhibit the expression of a gene foundin cancerous cells but not in normal cells, the therapeutic results areenhanced. Cancers which may be treated in accordance with the presentinvention would be numerous, with leukemias being one prominent example.

The formulations of the present invention also enhance the incorporationof oligos in the liposomes, as compared to prior art liposomalformulations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are photographs showing the uptake of (A) free and (B)liposomal pE oligos by ALL-1 cells.

FIG. 2A shows the extent of growth inhibition that resulted when ALL-1and HL60 cells were incubated with liposomal-pE antisense oligostargeted against the B1/A2 breakpoint junction of Bcr-Abl mRNA.

FIG. 2B shows the extent of growth inhibition that resulted when ALL-1cells were incubated with B1/A2 liposomal-pE antisense oligos and B2/A2liposomal-pE control oligos.

FIG. 3A shows the extent of growth inhibition that resulted when BV173and HL60 cells were incubated with liposomal-pE antisense oligostargeting against the B2/A2 breakpoint junction of Bcr-Abl mRNA.

FIG. 3B shows the extent of growth inhibition that resulted when BV173cells were incubated with liposomal-pE antisense oligos targetingagainst the B2/A2 breakpoint junction and with control oligos targetingagainst the B1/A2 breakpoint junction.

FIG. 4A shows the extent of growth inhibition that resulted when K562and HL60 cells were incubated with liposomal-pE antisense oligostargeting against the B3/A2 breakpoint junction of Bcr-Abl MRNA.

FIG. 4B shows the extent of growth inhibition that resulted when K562cells were incubated with liposomal-pE antisense oligos targetingagainst the B3/A2 breakpoint junction and with control oligos targetingagainst the B1/A2 breakpoint junction.

FIG. 5 shows the extent of growth inhibition that resulted when Tween20-containing liposomal-pE oligos were incubated with BV173 cells.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

"Liposomes" is used in this patent to mean lipid-containing vesicleshaving a lipid bilayer, as well as other lipid carrier particles whichcan entrap antisense oligonucleotides. The liposomes can be made of oneor more phospholipids, as long as the lipid material is substantiallyuncharged. It is important that the composition be substantially free ofanionic and cationic phospholipids and cholesterol. Suitablephospholipids include phosphatidyl cholines and others that are wellknown to persons that are skilled in this field. The liposomes can be,for example, unilamellar, multilamellar, or have an undefined lamellarstructure. A pharmaceutical composition comprising the liposomes willusually include a sterile, pharmaceutically acceptable carrier ordiluent, such as water or saline solution.

"Entrap," "encapsulate," and "incorporate" are used in this patent tomean that the oligo is enclosed within at least some portion of theinner aqueous space (including the interlamellar regions of the bilayer)of the liposome.

A composition of the present invention is preferably administered to apatient parenterally, for example by intravenous, intraarterial,intramuscular, intralymphatic, intraperitoneal, subcutaneous,intrapleural, or intrathecal injection, or may be used in ex vivo bonemarrow purging. Preferred dosages are between 5-25 mg/kg. Theadministration is preferably repeated on a timed schedule until thecancer disappears or regresses, and may be in conjunction with otherforms of therapy.

The making and use of the present invention is further illustrated bythe following examples.

EXAMPLE 1

Materials

Phosphodiester and phosphorothioate oligonucleotides were provided byGenta Incorporated. Phospholipids were purchased from Avanti PolarLipids.

Oligonucleotide Labeling

Phosphodiesters were labeled at 37° C. for 8 h with ³² Pγ!ATP at the 5'end by T4 kinase. The labeled oligonucleotide was precipitated withethanol at -20° C. overnight. After washing with 70% ethanol threetimes, phosphodiester oligonucleotides were twice filtered with aMicrocon-3 filter to separate the labeled oligonucleotides from free ³²Pγ!ATP.

Phosphorothioates, labeled with ³⁵ S, were provided by GentaIncorporated.

Liposomal-phosphodiester Preparation

Phosphodiester oligonucleotides dissolved in distilled water were mixedwith phospholipids in the presence of excess t-butanol so that the finalvolume of t-butanol in the mixture was 80-90%. Trace amounts of ³H!cholestanyl ether and ³² P!phosphodiester were also added to themixture as lipid and oligonucleotide markers, respectively. The mixturewas vortexed before being frozen in an acetone/dry ice bath. The frozenmixture was lyophilized and hydrated with hepes buffered saline (1 mMHepes and 10 mM NaCl) overnight. Liposomes were twice sonicated for 10min in a bath type sonicator.

Liposomal-phosphorothioate Preparation

Liposomal-phosphorothioates were prepared similarly to that describedfor liposomal-phosphodiesters, except phosphorothioates, instead ofphosphodiesters, were used. Also, ³⁵ S!phosphorothioates, instead of ³²P!phosphodiesters, were used as the oligonucleotide marker.

Separation of Free Oligonucleotides from those Incorporated in Liposomes

The separation of free phosphodiester or phosphorothioateoligonucleotides from those incorporated in liposomes was done bydialyzing the mixture against an excess 2500-fold volume of RPMI mediumat room temperature overnight. Aliquots of the preparation were takenbefore and after dialysis for liquid scintillation counting to assessthe incorporation of phosphodiester or phosphorothioate oligonucleotidesin liposomes.

Development of liposomal-phosphodiesters

Dioleoylphosphatidylcholine (DOPC) lipids were chosen for thephosphodiester (PD) incorporation because they are neutral while PD isnegatively charged. By using this lipid, electrostatic repulsion (whichcan lower incorporation) may be reduced. Positively charged lipids werenot used because they may induce non-specific cellular toxicity. Theinitial attempts of incorporating PD oligonucleotides into liposomeswere done by using freeze-and-thaw, and dehydration-rehydration methods.

(A) Freeze-and-thaw (FT) Method.

³ H!labeled DOPC lipids were evaporated from their organic solvent undernitrogen gas to form a lipid film. After vacuum desiccation, the lipidfilm was hydrated with hepes buffered saline (1 mM Hepes, 10 mM NaCl, pH8.0) and sonicated in a bath type sonicator. These pre-formed liposomeswere then mixed with ³² P!labeled PD oligonucleotides at a 100 or 1000to 1 molar ratio. The whole mixture was frozen in an acetone/dry icebath for 5-10 minutes and thawed at room temperature for 25-30 minutes.This process was repeated three times before the sample was loaded on aBioGel A0.5M column for separation of free PD oligonucleotides fromliposomal-PD. Aliquots were taken before and after the freeze-thawprocedure and were sent to liquid scintillation counting to determinethe incorporation of phosphodiesters into liposomes. Incorporation wasdetermined by ##EQU1## (B) Dehydration-Rehydration (DR) Method

³ H!labeled DOPC was evaporated from the organic solvent under nitrogengas to form a lipid film. After vacuum desiccation, the lipid film washydrated with hepes buffered saline and sonicated in a bath typesonicator. These pre-formed liposomes were then frozen in an acetone-dryice bath and lyophilized. The dried lipids were then rehydrated withdistilled water containing ³² P!phosphodiester oligonucleotides. Themolar ratio of DOPC to PD was either 100/1 or 1000/1. The mixture wasloaded on a BioGel A0.5M column for separation of free PDoligonucleotides from liposomal-PD. Aliquots were taken before and aftercolumn loading and were sent to liquid scintillation counting. Theincorporation was <5% (Table 1).

                  TABLE 1    ______________________________________    Comparison of Freeze-thaw (FT) and Dehydration-rehydration (DR)    methods on the incorporation of PD into DOPC liposomes.    Molar ratio   Methods  % incorporation    ______________________________________     100/1        FT       0                  DR       2.0    1000/1        FT       0                  DR       4.6    ______________________________________

With 500/1 and 5000/1 molar ratios of DOPC to PD, 0 and 5.2%incorporation were obtained, respectively.

It was then found that by adding t-butanol to the mixture beforefreezing and lyophilization, incorporation was raised to 11.9%. Also, byreducing the volume of distilled water added during the rehydrationprocess from 200 to 50 μl, 11.9 versus 1.8% incorporation was obtainedwith 1000/1 molar ratio. Effect of molar ratio on the incorporation wasagain measured with these improved conditions (Table 2).

                  TABLE 2    ______________________________________    Effect of lipid to oligonucleotides molar ratio    on the incorporation of PD into liposomes.    Molar ratio   % incorporation    ______________________________________    1000/1        11.9    2000/1        17.0    3000/1        13.4    4000/1        5.5    5000/1        9.2    ______________________________________

PD dissolved in distilled water was mixed with DOPC in the presence ofexcess t-butanol so that the final volume of t-butanol in the mixturewas 80-90% before being frozen in an acetone/dry ice bath andlyophilized. This was to avoid the step of preparing pre-formedliposomes. With this procedure, similar level of incorporation (16.2%)was obtained at the 2000/1 molar ratio.

It was decided to change the method of separation of free PD fromliposomal-PD because the recovery of lipids and PD were <50%. Two otherseparation methods were used: Microcon-10 filters and dialysis (Table3).

                  TABLE 3    ______________________________________    Comparison of different methods of separating free    PD from PD incorporated in liposomes..sup.a    Method    % Incorporation                          % Lipid and PD recovery    ______________________________________    Column    11.9        40-50    Filter    44.7        60-70    Dialysis  76.7        >90    ______________________________________     .sup.a DOPC was used to incorporate PD oligonucleotides at a 1000/1 molar     ratio.

When the liposomal mixture was sonicated for 10 min before the dialysisseparation process, similar level of incorporation was obtained. Anotherlipid, dimyristoyl phosphatidylcholine, was also used for PDincorporation. There was >85% incorporation.

Development of liposomal-phosphorothioates

Similar incorporation protocol was used with phosphorothioates (PT)since PT and PD are structural analogs. Various molar ratios of DOPC toPT were used (Table 4). The effect of sonication of the liposomalmixture (before dialysis) was also studied.

                  TABLE 4    ______________________________________    Effect of lipid to oligonucleotide molar ratios on    the incorporation of PT into liposomes.                 % incorporation    Molar ration without sonication                              with sonication    ______________________________________     10/1        >90          >90     50/1        >90          >90     100/1       45.8         55.5     200/1       44.1         49.1     500/1       27.8         47.0    1000/1       25.1         42.1    ______________________________________

EXAMPLE 2

Incorporation of p-ethoxy oligos into liposomes

pE oligos were purchased from Oligos Therapeutics (Willsonville, Oreg.).Phospholipids were purchased from Avanti Polar Lipids, Inc. (Alabaster,Ala.).

(a) Oligo labeling

pE oligos were labeled at 37° C. for 24 h with ³² Pγ!ATP at the 5' endby T4 polynucleotide kinase, and then precipitated with ethanol at -20°C. overnight. They were then washed with 70% ethanol three times toseparate the labeled oligo from free ³² Pγ!ATP.

(b) Liposome preparation

pE oligos dissolved in distilled H₂ O were mixed with phospholipids atvarious molar ratios in the presence of excess t-butanol so that thefinal volume of t-butanol in the mixture was at least 95%. Trace amountsof ³ H!cholestanyl ether and ³² P!pEs were also added to the mixture aslipid and oligo markers, respectively. The mixture was vortexed, frozenin an acetone/dry ice bath and then lyophilized. The lyophilizedpreparation was hydrated with Hepes buffered saline (1 mM Hepes and 10mM NaCl) at a final oligo concentration of 10-100 μM. Theliposomal-p-ethoxy oligos were sonicated for 10-20 min in a bath typesonicator.

(c) Separation of free pE oligos from those incorporated in liposomes

The separation of free pE oligos from those incorporated in liposomeswas done by dialyzing (MW cutoff=12-14,000) against 1000-fold excess ofHepes buffered saline at room temperature overnight. Aliquots ofliposomal-pE oligos were taken before and after dialysis for liquidscintillation counting to access the incorporation of pE oligos inliposomes.

(d) Incorporation efficiency

The lipid phosphatidylcholine (PC) was chosen for the incorporation ofpE oligos because both PC and pE oligos are neutral molecules so theyshould be compatible. Among all the different PCS, dioleoyl PC (DOPC)was chosen because it has a chain-melting phase transition temperatureat -15° to -20° C. Thus, at room temperature, DOPC is in the liquidcrystalline phase which is the ideal phase to prepare liposomes.

To incorporate pE oligos into liposomes, different molar ratios of pEoligos were mixed with DOPC together in the presence of excess oft-butanol. Trace amounts of radio labeled pE oligos and DOPC wereincluded in the mixture. The DOPC/pE oligos mixtures were frozen in adry ice/acetone bath before being lyophilized. The lyophilized DOPC/pEoligo powder was then hydrated with Hepes buffered saline so that thefinal oligo concentration was 10 μM. pE oligos were successfullyincorporated into DOPC liposomes, ranging between 28 to 83% efficiency(Table 5). The incorporation efficiency was dependent on the molarratios of DOPC to pE oligos: 10>100>5>1000:1.

                  TABLE 5    ______________________________________    Effect of molar ratio of DOPC to pE    oligos on the incorporation of pE oligos    Molar ratio of Incorporation    DOPC:pE oligos efficiency (%).sup.a    ______________________________________      5:1          45     10:1          83     100:1         71    1000:1         28    ______________________________________     .sup.a The incorporation efficiency values were obtained from three     separate experiments.

Delivery of pE oligos to leukemic cells

After achieving a high incorporation efficiency (>80%) of pE oligos intoliposomes, we then proceeded to test whether these liposomal-pE oligoscan reach the cytoplasm in which the mRNA is located. We had purchased a16-mer pE oligo labeled with rhodamine at the 5' end so that we couldvisualize the localization of the pE oligos by fluorescent microscopy.

(a) Incubation of pE oligos with leukemic cells

ALL-1 cells, which are human acute lymphocytic leukemic cells, wereused. Fifty thousand ALL-1 cells/well were plated in a 24-well plate in0.3 mL of medium. After 2 h of plating, final concentrations of 16 μM ofliposomal or free pE oligos conjugated with rhodamine were added toALL-1 cells. After 24 h of incubation, the cells were thrice washed withphosphate buffered saline before being viewed under a confocal laserscanning microscope. See FIG. 1 (Uptake of (A) free or (B) liposomal-pEoligos by ALL-1 cells).

Our data indicates that when incorporated into liposomes, higher amountsof pE oligos were taken up by the ALL-1 cells. The liposomes were ableto deliver the pE oligos to the cytoplasm.

Growth inhibition of liposomal-pE oligos on leukemic cells

We then proceeded to test whether liposomal-pE oligos can specificallyinhibit the growth of leukemic cells. We have used three different kindsof human leukemic cell lines: ALL-1 (acute lymphocytic leukemia), BV173and K562 (both are chronic myelogenous leukemia). All three cell linescontain the rearranged Philadelphia (Ph) chromosome which arises from areciprocal translocation of chromosomes 9 and 22. This translocationresults in the relocation of the c-Abl protooncogene from chromosome 9onto the 3' end of the breakpoint cluster region (Bcr) of chromosome 22,thus producing a hybrid Bcr-Abl gene. The breakpoint junctions where theBcr and the Abl genes fuse are different in the three cell lines. InALL-1 cells, the breakpoint junction is Bcr exon 1/Abl exon 2. In BV173cells, the breakpoint junction is Bcr exon 2/Abl exon 2. In K562 cells,the breakpoint junction is Bcr exon 3/Abl exon 2. All these hybrid genesproduce a novel Bcr-Abl fusion protein, which has enhanced tyrosinekinase activity that has been linked to the pathogenesis of theleukemias. Thus, inhibition of the production of the Bcr-Abl protein maylead to leukemic cell growth inhibition and even cell death. Tospecifically inhibit the production of the Bcr-Abl protein, we havedecided to target the antisense sequences against the breakpointjunctions of the Bcr-Abl MRNA which is only found in Phchromosome-positive leukemic cells, but not normal cells. This way, wehope to induce minimal non-specific toxic side effects as only leukemic,not normal, cell growth will be affected.

(a) Sequences of the pE antisense oligos (written from 5' to 3' end)antisense against Bcr exon 1/ Abl exon 2 (B1/A2) found in ALL-1 cells

    GAAGGGCTTCTGCGTC

antisense against Bcr exon 2/ Abl exon 2 (B2/A2) found in BV173 cells

    CTGAAGGGCTTCTTCC

antisense against Bcr exon 3/ Abl exon 2 (B3/A2) found in K562 cells

    GGGCTTTTGAACTCTGCT

(b) Delivery of liposomal-pE oligos to leukemic cells

Ten thousand ALL-1 or BV173 cells or five thousand K562 cells wereplated per well in a 96-well plate in 100 μL of RPMI medium containing10% fetal calf serum. After 2 h of plating, final concentrations of 0-10μM of liposomal-pE oligos were added to leukemic cells. The cells wereincubated with liposomal-pE oligos for 5 days. HL60 cells, a humanpromyelocytic cell line which does not have the Philadelphia chromosome,were used as control cells. They were plated under the same conditionsat ten thousand cells/well.

(c) Determination of the viability of the leukemic cells

At the end of the incubation, 100 μL of medium were added to each wellwhich makes the final volume of each well to be 200 μL. Then 50 μL ofcells were aliquoted and added to 96-well plates containing 130 μL ofmedium and 20 μL of alamarBlue dye. The cells will be incubated for 4-8more hours at 37° C. before being read directly on a microplate reader(Molecular Devices, Menlo Park, Calif.) at 570 and 595 nm. ThealamarBlue dye incorporates an oxidation-reduction indicator thatchanges color in response to chemical reduction of growth mediumresulting from cell growth. The difference in absorbance between 570 and595 nm will be taken as the overall absorbance value of the leukemiccells. The viabilities of leukemic cells treated with liposomal-pEoligos will be compared with those of the control untreated cells.

When ALL-1 and HL60 cells were incubated with liposomal-pE antisenseoligos targeted against the B1/A2 breakpoint junction of Bcr-Abl MRNA, adose-dependent growth inhibition of ALL-1, but not HL60, cells wasobserved (FIG. 2A). Similarly growth inhibition was observed with BV173and K562 cells when they were incubated with liposomal-pE antisenseoligos targeting against the B2/A2 and B3/A2 breakpoint junctions ofBcr-Abl mRNA, respectively (FIG. 3A, 4A). Under identical conditions,HL60 cells were not growth-inhibited.

To ensure that the growth inhibitory effects were sequence-dependent,the Ph chromosome-positive cell lines were incubated with antisense andcontrol liposomal-pE oligos. When ALL-1 cells were incubated with theB1/A2 liposomal-pE antisense oligos and the B2/A2 liposomal-pE controloligos, growth inhibition was induced (FIG. 2B). However, the B1/A2antisense oligos induced a much greater inhibitory effect. Similarly,higher inhibitory effects on BV173 and K562 cells were found with thecorresponding liposomal-pE antisense oligos than with the control oligos(FIG. 3B, 4B).

We also found that by including the detergent Tween 20 in theliposomal-pE oligo mixture, the potency of the inhibitory effects of theliposomal-pE oligos was increased. We added Tween 20 at 5% (wt. of pEoligos) in the liposomal-pE oligo mixture. Then the mixture wasvortexed, frozen in an acetone/dry ice bath before being lyophilized.The dried mixture was then hydrated and sonicated as stated previously.When Tween 20-containing liposomal-pE oligos were added to BV173 cells,100% growth inhibition was observed at 5 μM (FIG. 5) whereas under thesame conditions, 100% growth inhibition with normal liposomal-pE oligos(no Tween 20) was observed at 10 μM instead.

The preceding description of specific embodiments of the presentinvention is not intended to be a complete list of every possibleembodiment of the invention. Persons skilled in this field willrecognize that modifications can be made to the specific embodimentsdescribed here that would be within the scope of the present invention.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 3    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    GAAGGGCTTCTGCGTC16    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    CTGAAGGGCTTCTTCC16    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    GGGCTTTTGAACTCTGCT18    __________________________________________________________________________

We claim:
 1. A liposomal composition of antisense oligonucleotides,including (a) a liposome which consists essentially of neutralphospholipids, and (b) an antisense oligonucleotide that is entrapped inthe liposome and is selected from the group consisting of phosphodiesteroligonucleotides, phosphorothioate oligonucleotides, and p-ethoxyoligonucleotides; where if the antisense oligonucleotide is aphosphodiester oligonucleotide, the molar ratio of phospholipid to oligois between about 100:1 and about 3,000:1; if the antisenseoligonucleotide is a phosphorothioate oligonucleotide, the molar ratioof phospholipid to oligo is between about 10:1 and about 50:1: and ifthe antisense oligonucleotide is a p-ethoxy oligonucleotide, the molarratio of phospholipid to oligo is between about 5:1 and about 100:1. 2.The composition of claim 1, where the phospholipids arephosphatidylcholines.
 3. The composition of claim 1, where thephospholipids are dioleoylphosphatidyl choline.
 4. The composition ofclaim 1, where the antisense oligonucleotide is a phosphodiesteroligonucleotide, and the molar ratio of phospholipid to oligo is betweenabout 100:1 and about 3,000:1.
 5. The composition of claim 1, where theantisense oligonucleotide is a phosphorothioate oligonucleotide, and themolar ratio of phospholipid to oligo is between about 10:1 and about50:1.
 6. The composition of claim 1, where the antisense oligonucleotideis a p-ethoxy oligonucleotide, and the molar ratio of phospholipid tooligo is between about 5:1 and about 100:1.
 7. The composition of claim1, where the antisense oligonucleotide is a p-ethoxy oligonucleotidehaving the sequence GAAGGGCTTCTGCGTC.
 8. The composition of claim 1,where the antisense oligonucleotide is a p-ethoxy oligonucleotide havingthe sequence CTGAAGGGCTTCTTCC.
 9. The composition of claim 1, where theantisense oligonucleotide is a p-ethoxy oligonucleotide having thesequence GGGCTTTTGAACTCTGCT.
 10. A method of preparing a liposomalcomposition of antisense oligonucleotides, including the steps of:(a)hydrating a lyophilized composition that consists essentially of neutralphospholipids and an antisense oligonucleotide that is selected from thegroup consisting of phosphodiester oligonucleotides, phosphorothioateoligonucleotides, and p-ethoxy oligonucleotides; where if the antisenseoligonucleotide is a phosphodiester oligonucleotide, the molar ratio ofphospholipid to oligo is between about 100:1 and about 3,000:1; if theantisense oligonucleotide is a phosphorothioate oligonucleotide, themolar ratio of phospholipid to oligo is between about 10:1 and about50:1; and if the antisense oligonucleotide is a p-ethoxyoligonucleotide, the molar ratio of phospholipid to oligo is betweenabout 5:1 and about 100:1; thereby forming an aqueous suspension whichincludes free oligonucleotide and liposomes entrapping oligonucleotide;and (b) separating the free oligonucleotide from the liposomes bydialysis.
 11. The method of claim 10, where the aqueous suspension issonicated before dialysis.
 12. The method of claim 10, where thephospholipids are phosphatidylcholines.
 13. The method of claim 10,where the phospholipids are dioleoylphosphatidyl choline.
 14. The methodof claim 10, where the antisense oligonucleotide is a phosphodiesteroligonucleotide, and the molar ratio of phospholipid to oligo is betweenabout 100:1 and about 3,000:1.
 15. The method of claim 10, where theantisense oligonucleotide is a phosphorothioate oligonucleotide, and themolar ratio of phospholipid to oligo is between about 10:1 and about50:1.
 16. The method of claim 10, where the antisense oligonucleotide isa p-ethoxy oligonucleotide, and the molar ratio of phospholipid to oligois between about 5:1 and about 100:1.
 17. The method of claim 10, wherethe antisense oligonucleotide is a p-ethoxy oligonucleotide having thesequence GAAGGGCTTCTGCGTC as set forth in SEQ ID NO:1.
 18. The method ofclaim 10, where the antisense oligonucleotide is a p-ethoxyoligonucleotide having the sequence CTGAAGGGCTTCTTCC as set forth in SEQID NO:2.
 19. The method of claim 10, where the antisense oligonucleotideis a p-ethoxy oligonucleotide having the sequence GGGCTTTTGAACTCTGCT asset forth in SEQ ID NO:3.